HS2 Enhancer Function Is Blocked by a Transcriptional Terminator Inserted between the Enhancer and the Promoter

The HS2 enhancer in the β-globin locus control region regulates transcription of the globin genes 10-50 kb away. How the HS2 enhancer acts over this distance is not clearly understood. Earlier studies show that in erythroid cells the HS2 enhancer initiates synthesis of intergenic RNAs from sites within and downstream of the enhancer, and the enhancer-initiated RNAs are transcribed through the intervening DNA into the cis-linked promoter and gene. To investigate the functional significance of the enhancer-initiated transcription, here we inserted the lac operator sequence in the intervening DNA between the HS2 enhancer and the ϵ-globin promoter in reporter plasmids and integrated the plasmids into erythroid K562 cells expressing the lac repressor protein. We found that the interposed lac operator/repressor complex blocked the elongation of enhancer-initiated transcription through the intervening DNA and drastically reduced HS2 enhancer function as measured by the level of mRNA synthesized from the ϵ-globin promoter. The results indicate that the tracking and transcription mechanism of the HS2 enhancer-assembled transcriptional machinery from the enhancer through the intervening DNA into the cis-linked promoter can mediate enhancer-promoter interaction over a long distance. The HS2 enhancer in the β-globin locus control region regulates transcription of the globin genes 10-50 kb away. How the HS2 enhancer acts over this distance is not clearly understood. Earlier studies show that in erythroid cells the HS2 enhancer initiates synthesis of intergenic RNAs from sites within and downstream of the enhancer, and the enhancer-initiated RNAs are transcribed through the intervening DNA into the cis-linked promoter and gene. To investigate the functional significance of the enhancer-initiated transcription, here we inserted the lac operator sequence in the intervening DNA between the HS2 enhancer and the ϵ-globin promoter in reporter plasmids and integrated the plasmids into erythroid K562 cells expressing the lac repressor protein. We found that the interposed lac operator/repressor complex blocked the elongation of enhancer-initiated transcription through the intervening DNA and drastically reduced HS2 enhancer function as measured by the level of mRNA synthesized from the ϵ-globin promoter. The results indicate that the tracking and transcription mechanism of the HS2 enhancer-assembled transcriptional machinery from the enhancer through the intervening DNA into the cis-linked promoter can mediate enhancer-promoter interaction over a long distance. The locus control region (LCR) 1The abbreviations used are: LCR, locus control region; lacO/R, lac operator/repressor; IPTG, isopropyl 1-thio-β-d-galactopyranoside; CAT, chloramphenical acetyl transferase; GFP, green fluorescent protein; RT, reverse transcription; RPA, RNase protection assay; FACS, fluorescence-activated cell sorter; EMSA, electrophoretic mobility shift assay; nt, nucleotides; pp, primer pair. of the human β-globin gene domain, defined by four erythroid specific DNase I hypersensitive sites (HS1, -2, -3, and -4) and a ubiquitous HS5 site (1Tuan D. Solomon W. Li Q. London I. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6384-6388Crossref PubMed Scopus (437) Google Scholar, 2Forrester W. Takegawa S. Papayannopoulou T. Stamatoyannopoulos G. Groudine M. Nucleic Acids Res. 1987; 15: 10159-10177Crossref PubMed Scopus (296) Google Scholar, 3Grosveld F. Van Assendelft G.B. Kollias G. 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Cell. 1987; Full Text PDF PubMed Scopus Google Scholar, M. J. U. G. D. T. Cell. 1987; Full Text PDF PubMed Scopus Google Scholar) and is inserted in the of a gene far downstream of the promoter U. PubMed Scopus Google Scholar). by complex in cells can by the complex with M. Cell. 1987; Full Text PDF PubMed Scopus Google Scholar, M. J. U. G. D. T. Cell. 1987; Full Text PDF PubMed Scopus Google Scholar, U. PubMed Scopus Google Scholar). We the which the sequence inserted between the HS2 enhancer and the DNA located downstream of the HS2 enhancer in the genome the ϵ-globin and the chloramphenical acetyl reporter gene. To the endogenous globin gene we the which kb of intervening DNA that is with the ϵ-globin promoter in the genome and the green fluorescent protein reporter gene. The plasmids and the plasmids that not the interposed sequence integrated into erythroid K562 cell that a level of the protein. 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In the endogenous genome of K562 cells, the transcriptional machinery by the HS2 enhancer activate the far downstream ϵ-globin promoter by a tracking and transcription mechanism the kb of intergenic DNA between HS2 and the ϵ-globin gene in the K562 genome is transcribed by in a with transcription of the globin genes S. D. Li D. Mol. Cell. Biol. PubMed Scopus Google Scholar, M. Genes Dev. PubMed Scopus Google Scholar, M. W.G. J. Cell. Full Text PDF PubMed Scopus Google Scholar). intergenic RNAs synthesized from sites within and downstream of HS2 and downstream sites kb from B. W. B. and D. the transcriptional in a to not long track and the kb of intervening DNA to reach the ϵ-globin promoter and activate synthesis of the ϵ-globin The tracking and transcription mechanism of the transcription machinery is with in that the function of is to and to the cis-linked promoters M. K. Mol. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, M. A. 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Cell. 10: Full Text Full Text PDF PubMed Scopus Google Scholar). that in erythroid cells expressing the adult β-globin gene the HS2 enhancer interact directly with the β-globin promoter by a looping is not the HS2 enhancer through the to with the far downstream β-globin promoter with and promoters in It is that HS2 enhancer function the tracking and the looping in a during erythroid cell by the tracking of long enhancer function Q. Peterson K. Trends Genet. 1999; 10: 403-408Abstract Full Text Full Text PDF Scopus (201) Google Scholar, D. S. K. Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google Scholar, T. 1998; PubMed Scopus Google Scholar), a tracking mechanism can between the enhancer and the promoter over long intervening In the endogenous genome of human erythroid cells, the functional significance of the machinery in activity to the embryonic ϵ-globin gene and the downstream adult β-globin gene to to transcriptional downstream of the endogenous HS2 to the tracking and transcription of the complex and the transcription of the far downstream ϵ- and β-globin genes during erythroid cell We M. and D. of the with